Corneal disease and injury are the second leading causes of vision loss affecting over 10 million people worldwide. One of the most devastating outcomes of corneal wound repair is the onset of corneal fibrosis leading to the formation of a scar. Corneal fibrosis is a vision threatening condition characterized by the presence of corneal myofibroblasts and improper deposition of extracellular matrix components, including type III collagen. Studies of human fibrosis have been limited by the unavailability of human tissue and a culture model that mimics the 3-dimensional (3-D) organization of a fibrotic cornea. We propose to develop a 3-D model using human corneal fibroblasts stimulated by a stable Vitamin C (VitC) derivative and TGF-21. Preliminary data indicate that fibroblasts grown under these conditions stratify to multiple layers, secrete copious amounts of type III collagen, and differentiate into myofibroblasts in a manner resembling corneas undergoing scar formation. The hypothesis that we propose to test is that TGF-21 treatment of VitC stimulated human corneal fibroblasts results in a culture model that mimics human corneal scar formation. We propose two specific aims to examine the following questions: First, does TGF-21 treatment of human corneal fibroblasts stimulated with VitC generate a scar-like matrix? Second, is a scar matrix secreted and assembled differently than a corneal matrix during development? Relevance to public health Corneal scarring is the ultimate end product of all corneal traumas and diseases. Creation of a 3-D model of a human corneal scar would allow the examination of the regulation of corneal scar formation and determination of how scar matrix is secreted and organized. This would in turn allow us to better understand corneal fibrosis and potentially ameliorate scarring. PUBLIC HEALTH SIGNIFICANCE: One of the most devastating outcomes of corneal wound repair is the formation of a scar. Studies of human scar formation have been limited by the unavailability of human tissue and culture models. We propose to generate a culture model of corneal scarring using human cells.